Reducing immune tolerance induced by PD-L1

ABSTRACT

The present disclosure relates to compositions and methods for reducing immune tolerance associated with CAR T cell therapy. Embodiments of the present disclosure include isolated nucleic acid sequence comprising a nucleic acid sequence that encodes modified programmed cell death protein 1 (PD-1) and a nucleic acid sequence that encodes chimeric antigen receptor (CAR).

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation application of Internationalapplication number PCT/CN2016/075061, filed Mar. 1, 2016, title“Reducing Immune Tolerance Induced by PD-L1,” which claims priority toU.S. Provisional Patent Application No. 62/126,804, filed on Mar. 2,2015, entitled “Modified Cell and Uses thereof,” which is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is SDS1.0005US Sequence_ST25.txt. The text file isabout 19 KB, was created on Aug. 5, 2013, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present disclosure relates to modified cells and users, inparticularly to compositions and methods for reducing immune toleranceassociated with CAR T cell therapy.

BACKGROUND

T cell therapies have demonstrated efficacy and curative potential fortreating cancers. However, their uses are limited by the presence of animmunosuppressive microenvironment. The immunosuppressivemicroenvironment includes immune tolerance induced by the interactionbetween programmed death-1 (PD-1) and PD-L1 ligand (PD-L1). PD-1 is anegative coregulatory receptor on T cells and antigen-presenting cells.The PD-L1 is expressed by several cell types (e.g., tumor cells andother tissue cells), and appears to be dynamically regulated by theimmune microenvironment. Therefore, there is a need to address immunetolerance induced by the PD-L1 as to improve efficacy of T celltherapies.

SUMMARY

Embodiments of the present disclosure relate to an isolated nucleic acidsequence comprising a nucleic acid sequence that encodes modifiedprogrammed cell death protein 1 (PD-1) and a nucleic acid sequence thatencodes chimeric antigen receptor (CAR). In certain embodiments, themodified PD-1 and the CAR are expressed as gene products that areseparate polypeptides.

In some embodiments, the CAR is specific for a tumor antigen that ispresent on a cancer cell, and wherein the cancer cell expresses PD-L1.

In some embodiments, the tumor antigen comprises HER2, CD19, CD20, CD22,Kappa or light chain, CD30, CD33, CD123, CD38, ROR1, ErbB3/4, EGFR,EGFRvIII, EphA2, FAP, carcinoembryonic antigen, EGP2, EGP40, mesothelin,TAG72, PSMA, NKG2D ligands, B7-H6, IL-13 receptor α 2, IL-11 receptor α,MUC1, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AIMAGE A1, HLA-A2 NY-ESO-1, PSC1, folate receptor-α, CD44v7/8, 8H9, NCAM,VEGF receptors, 514, Fetal AchR, NKG2D ligands, CD44v6, TEM1, TEM8, orviral-associated antigens expressed by the tumor.

In some embodiments, the nucleic acid sequence that encodes the modifiedPD-1 comprises substitution or deletion of one or more nucleotides ascompared to a nucleic acid sequence encoding an intracellular part ofwild-type PD-1.

In some embodiments, the nucleic acid sequence that encodes the modifiedPD-1 comprises deletion of multiple nucleotides as compared to a nucleicacid sequence encoding an intracellular part of wild-type PD-1. Incertain embodiments, the modified PD-1 comprises the nucleic acidsequence of SEQ ID NO: 14.

In some embodiments, the nucleic acid sequence that encodes the modifiedPD-1 comprises a nucleic acid encoding a truncated PD-1 that does notinclude an intracellular domain. In certain embodiments, the nucleicacid sequence that encodes the modified PD-1 comprises the nucleic acidsequence of SEQ ID NO: 12.

In some embodiments, the modified PD-1 comprises one or more pointmutations as compared to wild-type PD-1. In certain embodiments, thepoint mutation comprises one or two amino acid point mutations ofphosphorylation sites of wild-type PD-1.

The embodiments further relate to an expression vector comprising thenucleic acid sequence as described above. In some embodiments, theexpression vector is a viral vector selected from the group consistingof a retroviral vector, lentiviral vector, adenoviral vector, and anadeno-associated viral vector.

The embodiments further relate to a cell comprising the expressionvector of the present disclosure. In some embodiments, the cell isselected from the group consisting of a T cell, NK cell, and a NKT cell.

The embodiments further relate to a pharmaceutical compositioncomprising an antitumor effective amount of a population of human Tcells, wherein the human T cells of the population include human T cellsthat comprises the isolated nucleic acid sequence as described above. Insome embodiments, an inhibitory effect of PD-L1 on cytokine productionof the human T cells of the population is less than an inhibitory effectof PD-L1 on cytokine production of human T cells that do not comprise atleast a part of the nucleic acid sequence that encodes the modifiedPD-1.

The embodiments further relate to a method of treating a cancer in ahuman patient, the method comprising administering to the human patientthe pharmaceutical composition as described in the present disclosure.

The embodiments further relate to a cell engineered to express modifiedPD-1 and chimeric antigen receptors (CAR), wherein the modified PD-1does not include a transmembrane part or an intracellular part of PD-1,or a combination thereof. In some embodiments, the cell expressessoluble PD-1 such as to disrupt PD-1 binding to PD-L-1. For example, thesoluble PD-1 is not attached to a cell membrane of the cell.

The embodiments further relate to a method of treating and/or inhibitingcancer of a subject. The method includes administering to the subject atherapeutically effective amount of a soluble receptor including anextracellular domain of PD-1. In some embodiments, the soluble receptorbinds a PD-L1 protein, and the soluble receptor disrupts PD-1 signalingof cancer cells and/or disrupts PD-1 binding to PD-L1. In certainembodiments, the isolated soluble receptor polypeptide includes aminoacid residues 20-519 of SEQ ID NO:9.

The embodiments further related to modified cell including a receptorpolypeptide, a cytoplasmic domain of the receptor polypeptide beingtruncated, the receptor polypeptide being at least one of a Programmedcell death protein 1 (PD-1) receptor polypeptide, cytotoxicT-lymphocyte-associated protein 4 (CTLA-4) receptor polypeptide, or B-and T-lymphocyte attenuator (BTLA) receptor.

The embodiments further relate to a method for treating a subject havinga disease. The method includes administering a cell to the subjecthaving the disease, wherein the cell is genetically modified to expressa receptor polypeptide, a cytoplasmic domain of the receptor polypeptidebeing truncated, the receptor polypeptide being at least one of aProgrammed cell death protein 1 (PD-1) receptor polypeptide, cytotoxicT-lymphocyte-associated protein 4 (CTLA-4) receptor polypeptide, or B-and T-lymphocyte attenuator (BTLA) receptor; and a chimeric antigenreceptor (CAR) including an antigen recognition domain of a specificantibody and an intracellular domain or a modified or wild-type T cellreceptor, the specific antibody binding to an antigen.

In some embodiments, the receptor polypeptide is the PD-1 receptorpolypeptide, and wherein the cytoplasmic domain of the PD-1 receptorpolypeptide contains an immunoreceptor tyrosine-based motif.

The embodiments further relate to modified cell including a reducedamount of one or more receptors as compared to a corresponding wild-typecell, the one or more receptors including at least one of a Programmedcell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4(CTLA-4), or B- and T-lymphocyte attenuator (BTLA).

The embodiments further relate to a method for treating a subject havinga disease. The method includes administering a cell to the subjecthaving the disease, wherein the cell is modified to express a reducedamount of one or more receptors as compared to a corresponding wild-typecell, the one or more receptors including at least one of a Programmedcell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4(CTLA-4), or B- and T-lymphocyte attenuator (BTLA).

In some embodiments, modified cell further includes a chimeric antigenreceptor (CAR) including an antigen recognition domain of a specificantibody and an intracellular domain, or a modified or wild-type T cellreceptor. In certain embodiments, the modified cell further includes achimeric antigen receptor (CAR) including an antigen recognition domainof a specific antibody and an intracellular domain or a modified orwild-type T cell receptor, the specific antibody binding to an antigen.

In some embodiments, the modified cell has reduced expression of one ormore genes associated with a biosynthesis pathway or transportationpathway of the one or more receptors as compared to the correspondingwild-type cell, or a combination thereof. In certain embodiments, themodified cell includes a disruption of the one or more genes. In certainembodiments, the modified cell includes a partial or a compete deletionof the one or more genes. In certain embodiments, the geneticallymodified cell replicate in vivo in the human patient.

In some embodiments, the modified cell form memory cells in the humanpatient. In these instances, the modified cells are administeredintravenously to the human patient. In certain embodiments, the modifiedcells persist in the human patient. For example, the modified cell is anautologous T cell. For another example, the cell may include at leastone of a B cell, a T cell, a NK cell, an embryotic cell, or a dendriticcell.

In some embodiments, the disease may include at least one of cancer,immune deficiencies, autoimmune disease, or obesity.

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

The Detailed Description is described with reference to the accompanyingfigures. The use of the same reference numbers in different figuresindicates similar or identical items.

FIG. 1 is a schematic diagram illustrating DNA constructs ofP0:CD19BBZETA, P1: CD19BBZETA-ires-soluble PD1,P2:CD19BBZETA-ires-Truncated PD1, P3: CD19BBZETA-ires-point mutationPD1, P4:CD19BBZETA-ires-Deletion PD1.

FIG. 2A is an image showing expression of CAR on transduced 293T andK562 cells.

FIG. 2B is an image showing expression of modified PD-1 on transduced293T and K562 cells.

FIG. 3A shows expression of CAR on transduced primary T cells.

FIG. 3B shows expression of modified PD-1 on transduced primary T cells.

FIG. 3C further shows expression of modified PD-1 on transduced primaryT cells.

FIG. 4A shows IFN-Gamma release by per 10⁴ T cells transduced with CARand modified PD-1s at the E:T ration of 10:1 of culturing with CD19⁺ for24 hours.

FIG. 4B demonstrates that CD19+ cells were killed by per 10⁴ T cellstransduced with CAR and modified PD-1s at the E:T ration of 10:1 ofculturing for 24 hours

FIG. 5 shows expression of PD-L1 on transduced NalM6 cells.

FIG. 6A shows IFN-gamma release of per 10⁴ T cells transduced with CARand modified PD-1s at the E:T ration of 10:1 of culturing with CD19⁺cells and CD19⁺/PD-L1⁺ cells for 24 hours. These result demonstrate thatInhibition of cytotoxicity induced by PD-L1 decreases on T cellstransduced with CAR and modified PD-1.

FIG. 6B illustrates loss of cytotoxicity induced by PD-L1 decrease on Tcells transduced with CAR and modified PD-1 as compared to T cellstransduced with CAR alone.

DETAILED DESCRIPTION

Overview

The present disclosure relates to the discovery that immune toleranceinduced by PD-L1 on CAR T cells based therapy can be reduced byexpression of genetically modified PD-1 on these T cells. In someembodiments, these T cells include a nucleic acid sequence that encodesCAR and genetically modified PD-1 such that the modified PD-1 and theCAR are expressed as gene products that are separate polypeptides onthese T cells. An example of the genetic modification includessubstitution or deletion of one or more nucleotides associated withexpression or a function of an intracellular part of PD-1.

In some embodiments, the present disclosure provides a T cell engineeredto express CAR against CD19 and modified PD-1 such that an inhibitoryeffect of PD-L1 on cytokine production of the T cell is significant lessthan an inhibitory effect of PD-L1 on cytokine production of a T cellthat do not include at least a part of the nucleic acid sequence thatencodes the modified PD-1. In some instances, the CAR is specific for atumor antigen that is present on a tumor cell, and the tumor cellexpresses PD-L1. Therefore, the engineered T cell of the presentdisclosure when infused into a patient can reduce immune toleranceinduced by PD-L1 of tumor cells and further eliminate these tumor cellsin vivo in the patient.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, preferred methods andmaterials are described. For the purposes of the present disclosure, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

The term “activation”, as used herein, refers to the state of a T cellthat has been sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), multi-specific antibodies (e.g., bispecific antibodies),and antibody fragments so long as they exhibit the desired biologicalactivity or function. The antibodies in the present disclosure may existin a variety of forms including, for example, polyclonal antibodies,monoclonal antibodies, Fv, Fab and F(ab)₂, as well as single chainantibodies and humanized antibodies (Harlow et al., 1999, In: UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, ColdSpring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426).

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region of the antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multi-specific antibodies formed from antibody fragments.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv including only three complementaritydetermining regions (CDRs) specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site. An “antibody heavy chain,” as used herein, refers to thelarger of the two types of polypeptide chains present in all antibodymolecules in their naturally occurring conformations. An “antibody lightchain,” as used herein, refers to the smaller of the two types ofpolypeptide chains present in all antibody molecules in their naturallyoccurring conformations. κ and λ light chains refer to the two majorantibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” as used herein is defined as a molecule that provokesan immune response, which may involve either antibody production, or theactivation of specific immunologically-competent cells, or both.Antigens may include any macromolecule, including virtually all proteinsor peptides, or molecules derived from recombinant or genomic DNA. Forexample, DNA including a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, anantigen need not be encoded solely by a full length nucleotide sequenceof a gene. Further, an antigen can be generated, synthesized or derivedfrom a biological sample including a tissue sample, a tumor sample, acell or a biological fluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect associated with a decrease in tumor volume, a decrease in thenumber of tumor cells, a decrease in the number of metastases, anincrease in life expectancy of a subject having tumor cells, oramelioration of various physiological symptoms associated with thecancerous condition. An “anti-tumor effect” can also be manifested bythe ability of the peptides, polynucleotides, cells and antibodies ofthe disclosure in prevention of the occurrence of tumor in the firstplace.

The term “auto-antigen” refers to an antigen mistakenly recognized bythe immune system as being foreign. Auto-antigens include cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autologous” is used to describe a material derived from thesame individual to which it is later to be re-introduced into theindividual.

“Allogeneic” is used to describe a graft derived from a different animalof the same species.

“Xenogeneic” is used to describe a graft derived from an animal of adifferent species.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include breast cancer,prostate cancer, ovarian cancer, cervical cancer, skin cancer,pancreatic cancer, colorectal cancer, renal cancer, liver cancer, braincancer, lymphoma, leukemia, lung cancer et al.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “includes” and “including” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of.” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

By “consisting essentially of” is meant including any elements listedafter the phrase, and limited to other elements that do not interferewith or contribute to the activity or action specified in the disclosurefor the listed elements. Thus, the phrase “consisting essentially of”indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands.

By “corresponds to” or “corresponding to” is meant (a) a polynucleotidehaving a nucleotide sequence that is substantially identical orcomplementary to all or a portion of a reference polynucleotide sequenceor encoding an amino acid sequence identical to an amino acid sequencein a peptide or protein; or (b) a peptide or polypeptide having an aminoacid sequence that is substantially identical to a sequence of aminoacids in a reference peptide or protein.

“Co-stimulatory ligand,” includes a molecule on an antigen presentingcell (e.g., an APC, dendritic cell, B cell, et al.) that specificallybinds a cognate co-stimulatory molecule on a T cell, thereby providing asignal which, in addition to the primary signal provided by, forinstance, binding of a TCR/CD3 complex with an MHC molecule loaded withpeptide, mediates a T cell response, including proliferation,activation, differentiation, et al. A co-stimulatory ligand can includeCD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, induciblecostimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM),CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin betareceptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that bindsToll ligand receptor and a ligand that specifically binds with B7-H3. Aco-stimulatory ligand also encompasses, inter alia, an antibody thatspecifically binds with a co-stimulatory molecule present on a T cell,such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such asproliferation. Co-stimulatory molecules include an MHC class I molecule,BTLA and a Toll-like receptor.

A “co-stimulatory signal” refers to a signal, which in combination witha primary signal, such as TCR/CD3 ligation, leads to T cellproliferation and/or upregulation or down regulation of key molecules.

As used herein, the terms “disease” and “condition” may be usedinterchangeably or may be different in that the particular malady orcondition may not have a known causative agent (so that etiology has notyet been worked out) and it is therefore not yet recognized as a diseasebut only as an undesirable condition or syndrome, wherein a more or lessspecific set of symptoms have been identified by clinicians. As usedherein, a “disease” is a state of health of a subject wherein thesubject cannot maintain homeostasis, and wherein if the disease is notameliorated then the subject's health continues to deteriorate. Incontrast, a “disorder” in a subject is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

As used herein, the term “effective” means adequate to accomplish adesired, expected, or intended result. For example, an “effectiveamount” may be an amount of a compound sufficient to produce atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

With regard to polynucleotides, the term “exogenous” refers to apolynucleotide sequence that does not naturally-occur in a wild-typecell or organism, but is typically introduced into the cell by molecularbiological techniques. Examples of exogenous polynucleotides includevectors, plasmids, and/or man-made nucleic acid constructs encoding adesired protein. With regard to polynucleotides, the term “endogenous”or “native” refers to naturally-occurring polynucleotide sequences thatmay be found in a given wild-type cell or organism. Also, a particularpolynucleotide sequence that is isolated from a first organism andtransferred to second organism by molecular biological techniques istypically considered an “exogenous” polynucleotide with respect to thesecond organism. In specific embodiments, polynucleotide sequences canbe “introduced” by molecular biological techniques into a microorganismthat already contains such a polynucleotide sequence, for instance, tocreate one or more additional copies of an otherwise naturally-occurringpolynucleotide sequence, and thereby facilitate overexpression of theencoded polypeptide.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector including a recombinantpolynucleotide including expression control sequences operatively linkedto a nucleotide sequence to be expressed. An expression vector includessufficient cis-acting elements for expression; other elements forexpression can be supplied by the host cell or in an in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and viruses(e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associatedviruses) that incorporate the recombinant polynucleotide.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” refers to a class of proteins, whichfunction as antibodies. The five members included in this class ofproteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibodythat is present in body secretions, such as saliva, tears, breast milk,gastrointestinal secretions and mucus secretions of the respiratory andgenitourinary tracts. IgG is the most common circulating antibody. IgMis the main immunoglobulin produced in the primary immune response inmost subjects. It is the most efficient immunoglobulin in agglutination,complement fixation, and other antibody responses, and is important indefense against bacteria and viruses. IgD is the immunoglobulin that hasno known antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide”, as used herein, refers to apolynucleotide, which has been purified from the sequences which flankit in a naturally-occurring state, e.g., a DNA fragment which has beenremoved from the sequences that are normally adjacent to the fragment.Alternatively, an “isolated peptide” or an “isolated polypeptide” andthe like, as used herein, refer to in vitro isolation and/orpurification of a peptide or polypeptide molecule from its naturalcellular environment, and from association with other components of thecell.

In the context of the present disclosure, the following abbreviationsfor the commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” et al. are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human. In some embodiments, the term “subject” is intended toinclude living organisms in which an immune response can be elicited(e.g., mammals). Examples of subjects include humans, dogs, cats, mice,rats, and transgenic species thereof.

The recitation “polynucleotide” or “nucleic acid” as used hereindesignates mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term typically refersto polymeric form of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNAand RNA.

The terms “polynucleotide variant” and “variant” and the like refer topolynucleotides displaying substantial sequence identity with areference polynucleotide sequence or polynucleotides that hybridize witha reference sequence under stringent conditions that are definedhereinafter. These terms also encompass polynucleotides that aredistinguished from a reference polynucleotide by the addition, deletionor substitution of at least one nucleotide. Accordingly, the terms“polynucleotide variant” and “variant” include polynucleotides in whichone or more nucleotides have been added or deleted, or replaced withdifferent nucleotides. In this regard, it is well understood in the artthat certain alterations inclusive of mutations, additions, deletionsand substitutions can be made to a reference polynucleotide whereby thealtered polynucleotide retains the biological function or activity ofthe reference polynucleotide, or has increased activity in relation tothe reference polynucleotide (i.e., optimized). Polynucleotide variantsinclude, for example, polynucleotides having at least 50% (and at least51% to at least 99% and all integer percentages in between, e.g., 90%,95%, or 98%) sequence identity with a reference polynucleotide sequencedescribed herein. The terms “polynucleotide variant” and “variant” alsoinclude naturally-occurring allelic variants and orthologs that encodethese enzymes.

“Polypeptide,” “polypeptide fragment,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues andto variants and synthetic analogues of the same. Thus, these terms applyto amino acid polymers in which one or more amino acid residues aresynthetic non-naturally occurring amino acids, such as a chemicalanalogue of a corresponding naturally occurring amino acid, as well asto naturally-occurring amino acid polymers. In certain aspects,polypeptides may include enzymatic polypeptides, or “enzymes”, whichtypically catalyze (i.e., increase the rate of) various chemicalreactions.

The recitation polypeptide “variant” refers to polypeptides that aredistinguished from a reference polypeptide sequence by the addition,deletion or substitution of at least one amino acid residue. In certainembodiments, a polypeptide variant is distinguished from a referencepolypeptide by one or more substitutions, which may be conservative ornon-conservative. In certain embodiments, the polypeptide variantcomprises conservative substitutions and, in this regard, it is wellunderstood in the art that some amino acids may be changed to otherswith broadly similar properties without changing the nature of theactivity of the polypeptide. Polypeptide variants also encompasspolypeptides in which one or more amino acids have been added ordeleted, or replaced with different amino acid residues.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence. The expression “control sequences” refers toDNA sequences necessary for the expression of an operably linked codingsequence in a particular host organism. The control sequences that aresuitable for prokaryotes, for example, include a promoter, optionally anoperator sequence, and a ribosome binding site. Eukaryotic cells areknown to utilize promoters, polyadenylation signals, and enhancers.

The term “bind,” “binds,” or “interacts with” means that one moleculerecognizes and adheres to a particular second molecule in a sample ororganism, but does not substantially recognize or adhere to otherstructurally unrelated molecules in the sample. By the term“specifically binds,” as used herein with respect to an antibody, ismeant an antibody which recognizes a specific antigen, but does notsubstantially recognize or bind other molecules in a sample. Forexample, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors may include additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis. Soluble receptor polypeptides are said to be substantiallyfree of transmembrane and intracellular polypeptide segments when theylack sufficient portions of these segments to provide membrane anchoringor signal transduction, respectively.

By “statistically significant,” it is meant that the result was unlikelyto have occurred by chance. Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which is the frequency or probability with whichthe observed event would occur, if the null hypothesis were true. If theobtained p-value is smaller than the significance level, then the nullhypothesis is rejected. In simple cases, the significance level isdefined at a p-value of 0.05 or less. A “decreased” or “reduced” or“lesser” amount is typically a “statistically significant” or aphysiologically significant amount, and may include a decrease that isabout 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100,500, 1000 times) (including all integers and decimal points in betweenand above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or leveldescribed herein.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such assignal transduction via the TCR/CD3 complex. Stimulation can mediatealtered expression of certain molecules, such as downregulation ofTGF-β, and/or reorganization of cytoskeletal structures, et al.

A “stimulatory molecule” refers to a molecule on a T cell thatspecifically binds with a cognate stimulatory ligand present on anantigen presenting cell.

A “stimulatory ligand” refers to a ligand that when present on anantigen presenting cell (e.g., an APC, a dendritic cell, a B-cell, etal.) can specifically bind with a cognate binding partner (referred toherein as a “stimulatory molecule”) on a T cell, thereby mediating aprimary response by the T cell, including activation, initiation of animmune response, proliferation, et al. Stimulatory ligands arewell-known in the art and encompass, inter alia, an MHC Class I moleculeloaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28antibody, and a superagonist anti-CD2 antibody.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which includes an isolated nucleicacid and which can be used to deliver the isolated nucleic acid to theinterior of a cell. Numerous vectors are known in the art includinglinear polynucleotides, polynucleotides associated with ionic oramphiphilic compounds, plasmids, and viruses. Thus, the term “vector”includes an autonomously replicating plasmid or a virus. The term shouldalso be construed to include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example,polylysine compounds, liposomes, et al. Examples of viral vectorsinclude, but are not limited to, adenoviral vectors, adeno-associatedvirus vectors, retroviral vectors, et al. For example, lentiviruses arecomplex retroviruses, which, in addition to the common retroviral genesgag, pol, and env, contain other genes with regulatory or structuralfunction. Lentiviral vectors are well known in the art. Some examples oflentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 andthe Simian Immunodeficiency Virus: SIV. Lentiviral vectors have beengenerated by multiply attenuating the HIV virulence genes, for example,the genes env, vif, vpr, vpu and nef are deleted making the vectorbiologically safe.

Ranges: throughout this disclosure, various aspects of the disclosurecan be presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

The present disclosure relates to isolated nucleic acid sequences,vectors including the isolated nucleic acid sequences, cells includingthe isolated nucleic acid sequences and methods of treating cancer usingthese cells.

Compositions and Therapeutic Application

Embodiments herein relate to an isolated nucleic acid sequence includinga nucleic acid sequence that encodes modified programmed cell deathprotein 1 (PD-1) and a nucleic acid sequence that encodes chimericantigen receptor (CAR). In some embodiments, the modified PD-1 and theCAR are expressed as gene products that are separate polypeptides. Inthese instances, the CAR is specific for a tumor antigen that is presenton a cancer cell, and the cancer cell expresses PD-L1.

CARs are molecules generally including an extracellular andintracellular domain. The extracellular domain includes atarget-specific binding element. The intracellular domain (e.g.,cytoplasmic domain) includes a costimulatory signaling region and a zetachain portion. The costimulatory signaling region refers to a portion ofthe CAR including the intracellular domain of a costimulatory molecule.Costimulatory molecules are cell surface molecules other than antigensreceptors or their ligands that are required for an efficient responseof lymphocytes to antigen.

Between the extracellular domain and the transmembrane domain of theCAR, there may be incorporated a spacer domain. As used herein, the term“spacer domain” generally means any oligo- or polypeptide that functionsto link the transmembrane domain to, either the extracellular domain or,the cytoplasmic domain in the polypeptide chain. A spacer domain mayinclude up to 300 amino acids, preferably 10 to 100 amino acids and mostpreferably 25 to 50 amino acids.

In some embodiments, the target-specific binding element of the CAR inthe present disclosure may recognize a tumor antigen. Tumor antigens areproteins that are produced by tumor cells that elicit an immuneresponse, particularly T-cell mediated immune responses. Tumor antigensare well known in the art and include, for example, a glioma-associatedantigen, carcinoembryonic antigen (CEA), (3-human chorionicgonadotropin, alphafetoprotein (AFP), lectin-reactive AFP,thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase,RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF,prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53,prostein, PSMA, Her2/neu, survivin and telomerase, prostate-carcinomatumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2,CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor andmesothelin.

In some embodiments, the tumor antigen includes HER2, CD19, CD20, CD22,Kappa or light chain, CD30, CD33, CD123, CD38, ROR1, ErbB3/4, EGFR,EGFRvIII, EphA2, FAP, carcinoembryonic antigen, EGP2, EGP40, mesothelin,TAG72, PSMA, NKG2D ligands, B7-H6, IL-13 receptor α 2, IL-11 receptor α,MUC1, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AIMAGE A1, HLA-A2 NY-ESO-1, PSC1, folate receptor-α, CD44v7/8, 8H9, NCAM,VEGF receptors, 5T4, Fetal AchR, NKG2D ligands, CD44v6, TEM1, TEM8, orviral-associated antigens expressed by the tumor.

In some embodiments, the antigen binding element of the CAR of thedisclosure targets CD19. In some instances, the antigen binding elementof the CAR of the disclosure includes anti-CD19 scFV including the aminoacid sequence set forth in SEQ ID NO: 1.

In some embodiments, the transmembrane domain of the CAR of thedisclosure includes the CD8 or CD9 transmembrane domain. In someembodiments, the intracellular domain of the CAR of the disclosureincludes the intracellular domain of 4-1BB (CD137) or CD 28. PD-1, amember of the CD28 family, is a type 1 transmembrane protein with theextracellular domain containing a single immunoglobulin (Ig)-likevariable domain and the intracellular domain containing animmunoreceptor tyrosine-based inhibitory motif. PD-1 is expressed onactivated T-cells, B lymphocytes, natural killer cells, dendritic cells,and activated monocytes. Interaction between PD-1 and its ligands PD-L1and PD-L2 leads to T-cell exhaustion, inactivation, and apoptosis.

Exhausted T lymphocytes lose the ability to produce proinflammatorycytokines including IL-2, tumor necrosis factor-α, and interferon-γ.Expression of PD-L1 by diverse tissues mediates peripheral immunetolerance, and activation of the PD-1/PD-L1 axis limits the tissuedamage after a sustained immune/inflammatory response. PD-1-expressingtumor-infiltrating lymphocytes are associated with an impaired antitumoreffect and upregulation of PD-1, and PD-L1 is associated with outcome inseveral tumor types.

The embodiments relate to an isolated nucleic acid sequence including anucleic acid sequence that encodes modified PD-1 and a nucleic acidsequence that encodes chimeric antigen receptor (CAR). In someembodiments, the nucleic acid sequence encoding modified PD-1 mayinclude substitution or deletion of one or more nucleotides as comparedto a nucleic acid sequence encoding an intracellular part of wild-typePD-1. In certain embodiments, the nucleic acid sequence that encodes themodified PD-1 includes deletion of one or more nucleotides as comparedto a nucleic acid sequence encoding an intracellular part of wild-typePD-1. For example, the modified PD-1 is human PD-1 that includes thenucleic acid sequence of SEQ ID NO: 14.

In some embodiments, the nucleic acid sequence that encodes the modifiedPD-1 includes a nucleic acid encoding a truncated PD-1 that does notinclude an intracellular domain. For example, the nucleic acid sequencethat encodes the modified PD-1 includes the nucleic acid sequence of SEQID NO: 12.

In some embodiments, the modified PD-1 includes one or more pointmutations as compared to wild-type PD-1. In certain embodiments, pointmutations may include one or two amino acid point mutations ofphosphorylation sites of wild-type PD-1. For example, the amino acidpoint mutations include a point mutation of Tyrosine residue 223 and/orTyrosine reside 248.

The embodiments of the present disclosure further relate to a DNAconstruct including sequences encoding a CAR and a modified PD-1. Insome embodiments, the CAR can include any combination of CD3-zeta, CD28,4-1BB, et al. For example, the CAR of the disclosure includes anti-CD19scFv, human CD8 hinge and transmembrane domain, and human 4-1BB andCD3zeta signaling domains. In one embodiment, the CAR of the disclosureincludes the nucleic acid sequence set forth in SEQ ID NO: 1.

In some embodiments, internal ribosome entry sites (IRES) elements areused to create multigene, or polycistronic, or messages. For example, anIRES element may link a nucleic acid sequence encoding CAR and a nucleicacid sequence encoding one of various modified PD-1 (See Table 1).

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The embodiments of the present disclosure further relate to vectors inwhich a DNA of the present disclosure is inserted. Vectors derived fromretroviruses such as the lentivirus are suitable tools to achievelong-term gene transfer since they allow long-term, stable integrationof a transgene and its propagation in daughter cells. Lentiviral vectorshave the added advantage over vectors derived from onco-retrovirusessuch as murine leukemia viruses in that they can transducenon-proliferating cells, such as hepatocytes. They also have the addedadvantage of low immunogenicity.

The expression of natural or synthetic nucleic acids encoding CARs andmodified PD-1 is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to one or morepromoters, and incorporating the construct into an expression vector.The vectors can be suitable for replication and integration eukaryotes.Typical cloning vectors contain transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the desired nucleic acid sequence.

Additional information related to expression synthetic nucleic acidsencoding CARs and modified PD-1s and gene transfer into mammalian cellsis provided in U.S. Pat. No. 8,906,682, incorporated by reference in itsentirety.

The embodiments further relate to genetically modified cells (e.g., Tcells) expressing a CAR and a modified PD-1. In some embodiments, adominant negative PD-1 is introduced in the T cells such that thedominated negative PD-1 inhibits wild-type PD-1 activity induced byPD-L1 of a tumor cell. In certain embodiments, the genetically modifiedT cells express non-functional PD-1. For example, the geneticallymodified T cells express PD-1 molecules not including intracellulardomain, not including both intracellular and transmembrane domains, orincluding a point mutation as described in the present disclosure.

In some embodiments, an inhibitory effect of PD-L1 of a tumor cell oncytokine production of the genetically modified T cells of the presentdisclosure is less than an inhibitory effect of PD-L1 on cytokineproduction of T cells that do not include at least a part of the nucleicacid sequence that encodes the modified PD-1. For example, an inhibitoryeffect of PD-L1 on cytokine production of the genetically modified Tcells of the present disclosure is sufficient to reduce at least 5%, atleast 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90% of aninhibitory effect of PD-L1 on cytokine production of T cells that do notinclude at least a part of the nucleic acid sequence that encodes themodified PD-1.

In some embodiment, the genetically modified T cells include a nucleicacid sequence that encodes the modified PD-1 having one or more pointmutations as compared to wild-type PD-1. For example, the point mutationcomprises one or two amino acid point mutations of phosphorylation sitesof wild-type PD-1. In certain embodiments, the genetically modified Tcells include the nucleic acid sequence that encodes the modified PD-1and includes deletion of one or more nucleotides as compared to anucleic acid sequence encoding an intracellular part of wild-type PD-1.For example, the nucleic acid sequence that encodes the modified PD-1comprises the nucleic acid sequence of SEQ ID NO: 12 or SEQ ID NO: 14.

The embodiments further relate to methods for treating a patient for anillness including administering to the patient an effective amount ofthe engineered cells of the present disclosure. Various illnesses can betreated according to the present methods including cancer, such asovarian carcinoma, breast carcinoma, colon carcinoma, glioblastomamultiforme, prostate carcinoma and leukemia. In some embodiments, themethod includes administering to a human patient a pharmaceuticalcomposition including an antitumor effective amount of a population ofhuman T cells, wherein the human T cells of the population include humanT cells that comprises the nucleic acid sequence as described in thepresent disclosure.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may include non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may include solid tumors. Types ofcancers to be treated with the CARs of the disclosure include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the engineeredcells of the present disclosure are used in the treatment of cancer. Incertain embodiments, the cells of the present disclosure are used in thetreatment of patients at risk for developing cancer. Thus, the presentdisclosure provides methods for the treatment or prevention of cancercomprising administering to a subject in need thereof, a therapeuticallyeffective amount of the engineered T cells of the present disclosure.

The engineered T cells of the present disclosure may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentdisclosure may include a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions mayinclude buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present disclosure are preferably formulated for intravenousadministration.

Pharmaceutical compositions of the present disclosure may beadministered in a manner appropriate to the disease to be treated (orprevented). The quantity and frequency of administration will bedetermined by such factors as the condition of the patient, and the typeand severity of the patient's disease, although appropriate dosages maybe determined by clinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “a tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentdisclosure to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent disclosure, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol, may select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i. v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent disclosure are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present disclosure are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present disclosure, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the present disclosure may beused in combination with chemotherapy, radiation, irrimunosuppressiveagents, such as cyclosporin, azathioprine, methotrexate, mycophenolate,and FK506, antibodies, or other immunoablative agents such as CAM PATH,anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p7056 kinase that is important for growth factor induced signaling(rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun73:316-321, 1991; Bierer et al., Curr. Opin. Immun 5:763-773, 1993;Isoniemi (supra)). In a further embodiment, the cell compositions of thepresent disclosure are administered to a patient in conjunction with(e.g., before, simultaneously or following) bone marrow transplantation,T cell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cellcompositions of the present disclosure are administered following B-cellablative therapy such as agents that react with CD20, e.g., Rituxan. Forexample, in one embodiment, subjects may undergo standard treatment withhigh dose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentdisclosure. In an additional embodiment, expanded cells are administeredbefore or following surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766, incorporated by reference inits entirety).

Additional information on the methods of cancer treatment using engineerT cells is provided in U.S. Pat. No. 8,906,682, incorporated byreference in its entirety.

The embodiments further relate to a method of treating and/or inhibitingcancer of a subject. The method includes administering to the subject atherapeutically effective amount of a soluble receptor including anextracellular domain of PD-1. In some embodiments, the soluble receptorbinds a PD-L1 protein, and the soluble receptor disrupts PD-1 signalingof cancer cells and/or disrupts PD-1 binding to PD-L1. In certainembodiments, the isolated soluble receptor polypeptide includes aminoacid residues 20-519 of SEQ ID NO:9.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors may include additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis. Soluble receptor polypeptides are said to be substantiallyfree of transmembrane and intracellular polypeptide segments when theylack sufficient portions of these segments to provide membrane anchoringor signal transduction, respectively.

The embodiments further related to modified cell including a receptorpolypeptide, a cytoplasmic domain of the receptor polypeptide beingtruncated, the receptor polypeptide being at least one of a Programmedcell death protein 1 (PD-1) receptor polypeptide, cytotoxicT-lymphocyte-associated protein 4 (CTLA-4) receptor polypeptide, or B-and T-lymphocyte attenuator (BTLA) receptor.

The embodiments further relate to a method for treating a subject havinga disease. The method includes administering a cell to the subjecthaving the disease, wherein the cell is genetically modified to expressa receptor polypeptide, a cytoplasmic domain of the receptor polypeptidebeing truncated, the receptor polypeptide being at least one of aProgrammed cell death protein 1 (PD-1) receptor polypeptide, cytotoxicT-lymphocyte-associated protein 4 (CTLA-4) receptor polypeptide, or B-and T-lymphocyte attenuator (BTLA) receptor; and a chimeric antigenreceptor (CAR) including an antigen recognition domain of a specificantibody and an intracellular domain or a modified or wild-type T cellreceptor, the specific antibody binding to an antigen.

In some embodiments, the receptor polypeptide is the PD-1 receptorpolypeptide, and wherein the cytoplasmic domain of the PD-1 receptorpolypeptide contains an immunoreceptor tyrosine-based motif.

The embodiments further relate to modified cell including a reducedamount of one or more receptors as compared to a corresponding wild-typecell, the one or more receptors including at least one of a Programmedcell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4(CTLA-4), or B- and T-lymphocyte attenuator (BTLA).

The embodiments further relate to a method for treating a subject havinga disease. The method includes administering a cell to the subjecthaving the disease, wherein the cell is modified to express a reducedamount of one or more receptors as compared to a corresponding wild-typecell, the one or more receptors including at least one of a Programmedcell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4(CTLA-4), or B- and T-lymphocyte attenuator (BTLA).

In some embodiments, modified cell further includes a chimeric antigenreceptor (CAR) including an antigen recognition domain of a specificantibody and an intracellular domain, or a modified or wild-type T cellreceptor. In certain embodiments, the modified cell further includes achimeric antigen receptor (CAR) including an antigen recognition domainof a specific antibody and an intracellular domain or a modified orwild-type T cell receptor, the specific antibody binding to an antigen.

In some embodiments, the modified cell has reduced expression of one ormore genes associated with a biosynthesis pathway or transportationpathway of the one or more receptors as compared to the correspondingwild-type cell, or a combination thereof. In certain embodiments, themodified cell includes a disruption of the one or more genes. In certainembodiments, the modified cell includes a partial or a compete deletionof the one or more genes. In certain embodiments, the geneticallymodified cell replicate in vivo in the human patient.

In some embodiments, the modified cell form memory cells in the humanpatient. In these instances, the modified cells are administeredintravenously to the human patient. In certain embodiments, the modifiedcells persist in the human patient. For example, the modified cell is anautologous T cell. For another example, the cell may include at leastone of a B cell, a T cell, a NK cell, an embryotic cell, or a dendriticcell.

In some embodiments, the disease may include at least one of cancer,immune deficiencies, autoimmune disease, or obesity.

EXAMPLES

The present disclosure is further described by reference to thefollowing examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified. Thus, the present disclosure should in no way be construed asbeing limited to the following examples, but rather, should be construedto encompass any and all variations which become evident as a result ofthe teaching provided herein.

Example 1 Expression of CAR and PD-1 on HEK293T & K562 Cells

Lentiviral vectors that encodes a CD19 CAR and a modified PD-1 separatedby the encephalomyocarditis virus internal ribosomal entry sequence weregenerated (see Chimeric Receptors Containing CD137 Signal TransductionDomains Mediate Enhanced Survival of T Cells and Increased AntileukemicEfficacy In Vivo Molecular Therapy vol. 17 no. 8, 1453-1464 August 2009incorporated herein by reference). Information of DNA constructsencoding CAR and the modified PD-1 is provided in FIG. 1 and Table 1. Asummary of these constructs and modules are listed in Tables 1-2.

TABLE 1 DNA constructs and encoding modules PD-1 PD-1 Trans- PD-1 PD-1Extra- mem- Intracellu- Signal cellular brane lar domain Ref ConstructsName Peptide domain domain or residues P0 P0: CD19BBZETA No No No No TP1 P1: CD19BBZETA Yes Yes NO No T-ires-soluble PD1 P2 P2: CD19BBZETA YesYes Yes Residues T-ires-Truncated PD1 P3 P3: CD19BBZETA Yes Yes Yes Yes:aa223, Y T-ires-point mutation Y to A; PD1 aa248, Y to A P4 P4:CD19BBZETA Yes Yes Yes Residues T-ires-Deletion PD1

HEK293T & K562 cells were transduced with lentiviral vectors.Flow-cytometry were acquisition was performed and analyzed to determineexpression of CAR and PD-1 in these cells. As shown in FIGS. 2A and 2B,both KECK293T and K562 cells expressed CARs (See Box in FIG. 2A) andPD-1s (See Box FIG. 2B).

HEK293T and K562 cells were obtained from American Type CultureCollection (ATCC; Manassas, Va.). Techniques related to cell cultures,construction of lentiviral vectors, and flow-cytometry may be found inChimeric Receptors Containing CD137 Signal Transduction Domains MediateEnhanced Survival of T Cells and Increased Antileukemic Efficacy In VivoMolecular Therapy vol. 17 no. 8, 1453-1464 August 2009 incorporatedherein by reference, which is incorporated herein by reference.

TABLE 2 Sequence identifiers for various constructs SEQ ID NO: #Identify SEQ ID NO: 1 P0 (nucleic acid sequence) SEQ ID NO: 2 P1(nucleic acid sequence) SEQ ID NO: 3 P2 (nucleic acid sequence) SEQ IDNO: 4 P3 (nucleic acid sequence) SEQ ID NO: 5 P4 (nucleic acid sequence)SEQ ID NO: 6 IRES element (nucleic acid sequence) SEQ ID NO: 7PDL1-IRES-EGFP (nucleic acid sequence) SEQ ID NO: 8 PD-1 Signal Peptide(amino acid sequence) SEQ ID NO: 9 PD-1 Extracellular domain (amino acidsequence) SEQ ID NO: 10 PD-1 Transmembrane domain (amino acid sequence)SEQ ID NO: 11 P1: added Hydrophilic segment SEQ ID NO: 12 P2: PD-1Intracellular domain residues (amino acid sequence) SEQ ID NO: 13 P3:PD-1 Intracellular domain residues (amino acid sequence) SEQ ID NO: 14P4: PD-1 Intracellular domain residues (amino acid sequence) SEQ ID NO:15 P2: Removed part of PD-1 Intracellular domain (amino acid sequence)SEQ ID NO: 16 P4: Removed part of PD-1 Intracellular domain (amino acidsequence)

Example 2 Expression of CAR and PD-1 on Primary T Cells

Primary T cells were obtained from patients. The obtained primary Tcells were transduced with lentiviral vectors. Flow-cytometry wereacquisition was performed and analyzed to determine expression of CARand PD-1 in primary T cells. As shown in FIGS. 3A, 3B, and 3C, primary Tcells expressed CARs (See Box in FIG. 3A) and PD-1s (See Box FIGS. 3Band 3C).

Techniques related to cell cultures, construction of lentiviral vectors,and flow-cytometry may be found in Control of large, established tumorxenografts with genetically retargeted human T cells containing CD28 andCD137 domains. 3360-3365 PNAS Mar. 3, 2009 vol. 106 no. 9, which isincorporated herein by reference.

Example 3 Cytotoxic T-Lymphocyte Assay

In this assay, the cytotoxicity on target cells (i.e., K562-CD19) ismeasured by comparing survival of target cells culturing with effectorcells (i.e., transduced T cells) relative to the survival of targetcells culturing with negative control cells (i.e., non-transduced Tcells). Target cells and effector cells or negative control cells werecultured for about 24 hours with a number ratio between the target cellsand effector cells or negative control cells being about 10:1. Survivalrates of target cells and IFN-gamma production of transduced T cells andnon-transduced T cells were measured. As shown in FIGS. 4A and 4B,transduced T cells containing nucleic acid sequences encoding CAR andvarious PD-1s are capable of releasing IFN-gamma and killing CD19 cells.All error bars are representative of standard deviation.

Techniques related to cell cultures, construction of cytotoxicT-lymphocyte assay may be found in Control of large, established tumorxenografts with genetically retargeted human T cells containing CD28 andCD137 domains. 3360-3365 PNAS Mar. 3, 2009 vol. 106 no. 9, which isincorporated herein by reference.

Example 4 Construction of PD-L1-Ires-EGFP Nalm-6 Cell Lines

Lentiviral vectors that encodes a PD-L1-ires-EGFP (SEQ ID NO: 7) weregenerated. Nalco-6 cells were transduced with lentiviral vectors T cellsan MOI of 30 or an MOI of 100. Flow-cytometry were acquisition wasperformed and analyzed to determine expression of CAR and PD-1 in thesecells. As shown in FIG. 5, NaIM-6 PD-L1 expressed PD-L1.

Nalco-6 cells were obtained from American Type Culture Collection (ATCC;Manassas, Va.). Techniques related to cell cultures, construction oflentiviral vectors, and flow-cytometry may be found in Treatment ofAdvanced Leukemia in Mice with mRNA Engineered T Cells_(o) HUMAN GENETHERAPY 22:1575-1586 (December 2011), which is incorporated herein byreference.

Example 5 Inhibition of Cytotoxicity Induced by PD-L1 Decreases on TCells Transduced with CAR and Modified PD-1

Target cells (i.e., NalM-6 expressing PD-L1 or NalM-6) and variouseffector cells (i.e., transduced T cells) or negative control cells(i.e., non-transduced T cells) were cultured for about 24 hours with anumber ratio between the target cells and effector cells or negativecontrol cells being about 10:1. IFN-gamma production of transduced Tcells were measured. As shown in FIGS. 6A and 6B, IFN-gamma productionsby CAR T cells were reduced, which indicated loss of cytotoxicity inresponse to expression of PD-L1 on NalM-6 cells. Further, as compared toCAT T cells without modified PD-1, loss of cytotoxicity induced by PD-L1decrease on CAR T cells transduced with modified PD-1. For example, lossof cytotoxicity of T cells transduced with CAR and PD-1 having pointmutation (i.e., P3) is about 22%, which is lower than 43% of CAR T cellswithout modified PD-1 (i.e., P0). This indicates that Inhibition ofcytotoxicity induced by PD-L1 decreases on T cells transduced with CARand modified PD-1. All error bars are representative of standarddeviation.

Techniques related to cell cultures, construction of cytotoxicT-lymphocyte assay may be found in Chimeric Receptors Containing CD137Signal Transduction Domains Mediate Enhanced Survival of T Cells andIncreased Antileukemic Efficacy in Vivo Molecular Therapy vol. 17 no. 8,1453-1464 August 2009, which is incorporated herein by reference.

Embodiments

Embodiment 1: An isolated nucleic acid sequence comprising a nucleicacid sequence that encodes modified programmed cell death protein 1(PD-1) and a nucleic acid sequence that encodes chimeric antigenreceptor (CAR), wherein the modified PD-1 and the CAR are expressed asgene products that are separate polypeptides.

Embodiment 2: The isolated nucleic acid sequence of embodiment 1,wherein the CAR is specific for a tumor antigen that is present on acancer cell, and wherein the cancer cell or a cell associated with thecancer cell expresses PD-L1.

Embodiment 3: The isolated nucleic acid sequence of embodiment 2,wherein the tumor antigen comprises CD19.

Embodiment 4: The isolated nucleic acid sequence of embodiment 1,wherein the nucleic acid sequence that encodes the modified PD-1comprises substitution or deletion of one or more nucleotides ascompared to a nucleic acid sequence encoding an intracellular part ofwild-type PD-1.

Embodiment 5: The isolated nucleic acid sequence of embodiment 1,wherein the nucleic acid sequence that encodes the modified PD-1comprises deletion of multiple nucleotides as compared to a nucleic acidsequence encoding an intracellular part of wild-type PD-1.

Embodiment 6: The isolated nucleic acid sequence of embodiment 5,wherein the modified PD-1 comprises the nucleic acid sequence of SEQ IDNO: 14.

Embodiment 7: The isolated nucleic acid sequence of embodiment 4,wherein the nucleic acid sequence that encodes the modified PD-1comprises a nucleic acid encoding a truncated PD-1 that does not includean intracellular domain.

Embodiment 8: The isolated nucleic acid sequence of embodiment 7,wherein the nucleic acid sequence that encodes the modified PD-1comprises the nucleic acid sequence of SEQ ID NO: 12.

Embodiment 9: The isolated nucleic acid sequence of embodiment 1,wherein the modified PD-1 comprises a point mutation as compared towild-type PD-1.

Embodiment 10: The isolated nucleic acid sequence of embodiment 9,wherein the point mutation comprises one or two amino acid pointmutations of phosphorylation sites of wild-type PD-1.

Embodiment 11: An expression vector comprising the isolated nucleic acidsequence of any of embodiment 1 to 10.

Embodiment 12: The expression vector of embodiment 11, wherein theexpression vector is a viral vector selected from the group consistingof a retroviral vector, lentiviral vector, adenoviral vector, and anadeno-associated viral vector.

Embodiment 13: A cell comprising the expression vector of embodiment 11.

Embodiment 14: The cell embodiment 13, wherein the cell is selected fromthe group consisting of a T cell, NK cell, and a NKT cell.

Embodiment 15: A pharmaceutical composition comprising an antitumoreffective amount of a population of human T cells, wherein the human Tcells of the population include human T cells that comprises theisolated nucleic acid sequence of any of embodiments 1 to 10.

Embodiment 16: The pharmaceutical composition of embodiment 15, whereinan inhibitory effect of PD-L1 on cytokine production of the human Tcells of the population is less than an inhibitory effect of PD-L1 oncytokine production of human T cells that do not comprise at least apart of the nucleic acid sequence that encodes the modified PD-1.

Embodiment 17: A method of treating a cancer in a human patient, themethod comprising administering to the human patient the pharmaceuticalcomposition of embodiment 16.

Embodiment 18: A cell engineered to express modified PD-1 and chimericantigen receptors (CAR), wherein the modified PD-1 does not include atransmembrane part or an intracellular part of PD-1, or a combinationthereof.

Embodiment 19: The cell of embodiment 18, wherein the cell expressessoluble PD-1 such as to disrupt PD-1 binding to PD-L-1.

Embodiment 20: The cell of embodiment 19, wherein the soluble PD-1 isnot attached to a cell membrane of the cell.

Embodiment 21: A modified cell comprising: a receptor polypeptide, acytoplasmic domain of the receptor polypeptide being truncated, thereceptor polypeptide being at least one of a Programmed cell deathprotein 1 (PD-1) receptor polypeptide, cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4) receptor polypeptide, or B- and T-lymphocyteattenuator (BTLA) receptor.

Embodiment 22: A method for treating a subject having a disease, themethod comprising: administering a cell to the subject having thedisease, wherein the cell is genetically modified to express: a receptorpolypeptide, a cytoplasmic domain of the receptor polypeptide beingtruncated, the receptor polypeptide being at least one of a Programmedcell death protein 1 (PD-1) receptor polypeptide, cytotoxicT-lymphocyte-associated protein 4 (CTLA-4) receptor polypeptide, or B-and T-lymphocyte attenuator (BTLA) receptor; and a chimeric antigenreceptor (CAR) comprising an antigen recognition domain of a specificantibody and an intracellular domain or a modified or wild-type T cellreceptor, the specific antibody binding to an antigen.

Embodiment 23: The modified cell or the method of embodiment 21 or 22,wherein the receptor polypeptide is the PD-1 receptor polypeptide, andwherein the cytoplasmic domain of the PD-1 receptor polypeptide containsan immunoreceptor tyrosine-based motif.

Embodiment 24: The modified cell or the method of embodiment 21 or 22,wherein the specific antibody binds to an antigen.

Embodiment 25: A modified cell comprising: a reduced amount of one ormore receptors as compared to a corresponding wild-type cell, the one ormore receptors comprising at least one of a Programmed cell deathprotein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4),or B- and T-lymphocyte attenuator (BTLA).

Embodiment 26: A method for treating a subject having a disease, themethod comprising: administering a cell to the subject having thedisease, wherein the cell is modified to express: a reduced amount ofone or more receptors as compared to a corresponding wild-type cell, theone or more receptors comprising at least one of a Programmed cell deathprotein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4),or B- and T-lymphocyte attenuator (BTLA).

Embodiment 27: The modified cell of embodiments 21 or 25, wherein themodified cell further comprises a chimeric antigen receptor (CAR)comprising an antigen recognition domain of a specific antibody and anintracellular domain, or a modified or wild-type T cell receptor.

Embodiment 28: The method of embodiments 22 or 26, wherein the modifiedcell further comprises a chimeric antigen receptor (CAR) comprising anantigen recognition domain of a specific antibody and an intracellulardomain or a modified or wild-type T cell receptor, the specific antibodybinding to an antigen.

Embodiment 29: The modified cell or the method of embodiments 25 or 26,wherein the modified cell has reduced expression of one or more genesassociated with a biosynthesis pathway or transportation pathway of theone or more receptors as compared to the corresponding wild-type cell,or a combination thereof.

Embodiment 30: The modified cell or the method of embodiments 25 or 26,wherein the modified cell comprises a disruption of the one or moregenes.

Embodiment 31: The modified cell or the method of embodiments 25 or 26,wherein the modified cell comprises a partial or a compete deletion ofPD-1 gene.

Embodiment 32: The method of embodiments 22 or 26, wherein thegenetically modified cell replicate in vivo in the human patient.

Embodiment 33: The method of embodiments 22 or 26, wherein the modifiedcell form memory cells in the human patient.

Embodiment 34: The method of embodiments 22 or 26, wherein the modifiedcells are administered intravenously to the human patient.

Embodiment 35: The method of embodiments 22 or 26, wherein the modifiedcells persist in the human patient.

Embodiment 36: The method of embodiments 32, wherein the modified cellis an autologous T cell.

Embodiment 37: The modified cell or the method of embodiments 21, 22, 25or 26, wherein the cell comprises at least one of a B cell, a T cell, aNK cell, an embryotic cell, or a dendritic cell.

Embodiment 38: The method of any of embodiments 22 or 26, wherein thedisease comprises at least one of cancer, immune deficiencies,autoimmune disease, or obesity.

Embodiment 39: A method of treating and/or inhibiting cancer of asubject, the method comprising administering to the subject atherapeutically effective amount of a soluble receptor comprising anextracellular domain of PD-1.

Embodiment 40: The method of embodiment 39, wherein the soluble receptorbinds a PD-L1 protein, and the soluble receptor disrupts PD-1 signalingof cancer cells and/or disrupts PD-1 binding to PD-L1.

Embodiment 41: The method of embodiment 39, wherein the soluble receptorpolypeptide includes amino acid residues 20-519 of SEQ ID NO:9.

What is claimed is:
 1. A pharmaceutical composition comprising apopulation of human T cells, wherein the population of human T cellsinclude human T cells that comprise a nucleic acid molecule comprising:a first nucleic acid sequence that encodes modified programmed celldeath protein 1 (PD-1), which comprises the amino acid sequence of SEQID NO: 13, and a second nucleic acid sequence that encodes a chimericantigen receptor (CAR) comprising: an extracellular domain thatrecognizes a tumor antigen, a transmembrane domain, and an intracellulardomain comprising a CD3-zeta signaling domain and one or more signalingdomains of one or more costimulatory molecules, wherein the modifiedPD-1 and the CAR are expressed as gene products that are separatepolypeptides.
 2. The pharmaceutical composition of claim 1, wherein aninhibitory effect induced by a target cell on cytokine production of thehuman T cells is reduced as compared to human T cells that express theCAR and lack the modified PD-1, and the target cell express the tumorantigen and programmed death ligand 1 (PD-L1).
 3. The pharmaceuticalcomposition of claim 1, wherein the tumor antigen is CD19.
 4. Thepharmaceutical composition of claim 1, wherein the one or morecostimulatory molecules include 4-1 BB.
 5. A method of reducing aninhibitory effect induced by a target cell on cytokine production ofhuman T cells that express a chimeric antigen receptor (CAR), the methodcomprising introducing a nucleic acid molecule into the human T cells,wherein: the nucleic acid molecule that encodes modified programmed celldeath protein 1 (PD-1), which comprises the amino acid sequence of SEQID NO: 13, the CAR comprising: an extracellular domain that recognizes atumor antigen, a transmembrane domain, and an intracellular domaincomprising a CD3-zeta domain and one or more signaling domains of one ormore costimulatory molecules, the target cell express the tumor antigenand programmed death ligand 1 (PD-L1), and the modified PD-1 and the CARare expressed as gene products that are separate polypeptides.
 6. Themethod of claim 4, wherein the tumor antigen is CD19.
 7. The method ofclaim 4, wherein the one or more costimulatory molecules include 4-1 BB.